A variety of techniques are currently available for efficient amplification of nucleic acids even from a few molecules of a starting nucleic acid template. These include polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), or rolling circle amplification (RCA). Many of these techniques involve an exponential amplification of the starting nucleic acid template, and generate a large number of amplified products in a short span of time.
Nucleic acid amplification techniques are often employed in nucleic acid-based assays used for analyte detection, sensing, forensic and diagnostic applications, genome sequencing, whole-genome amplification, and the like. Such applications often require amplification techniques having high specificity, sensitivity, accuracy, and robustness. However, most of the currently available techniques for nucleic acid amplification suffer from high background signals, which are generated by non-specific amplification reactions yielding undesired/false amplification products. These non-specific amplification reactions hinder effective utilization of many of these techniques in critical nucleic acid-based assays. For example, if such an amplification reaction were used for diagnostic applications, a false-positive amplification (e.g., formation of amplification products even when the template nucleic acid is absent) may likely result in a wrong diagnosis. Such non-specific, background amplification reactions become even more problematic where the target nucleic acid to be amplified is available only in limited quantities (e.g., whole-genome amplification from a single DNA molecule).
Non-specific, background amplification reactions may be due to exogenous, non-target amplification (e.g., amplification of a contaminating nucleic acid), amplification of untargeted sequences, or primer amplification (endogenous factors). A frequent source of non-specific amplification in a nucleic acid amplification reaction results from various primer gymnastics. A primer may hybridize to regions of a nucleic acid (either in a target nucleic acid itself or in a contaminating nucleic acid) that share some homology with a targeted sequence of the target nucleic acid. If the 3′ end of a primer has sufficient homology to an untargeted region, the untargeted region may get amplified. Non-specific amplification may also result from nucleic acid template-independent primer-primer interactions. Primers may form primer-dimer structures by intra- or inter-strand primer annealing (intra molecular or inter molecular hybridizations), and may get amplified. The resultant spurious primer extension products may further get amplified, and may sometimes predominate, inhibit, or mask the amplification of the targeted sequence. In addition, during amplification reaction, the amplification products may self-hybridize, allowing the nucleic acid polymerase to generate hybrid products or chimeric products.
Random primers (e.g., N6, where N=A/T/G/C) are often used for nucleic acid amplification that demands amplification without significant sequence bias. They are useful for applications such as whole-genome amplification, or for amplification of a target nucleic acid with unknown sequence. However, such random primers are also most susceptible for primer-dimer structure formation, and thus lead to higher levels of non-specific, endogenous background amplification. Hence, the use of random primers in high efficiency nucleic acid amplification techniques is often problematic. Constrained-randomized primers that cannot cross-hybridize via intra- or inter-molecular hybridization (e.g., R6, where R=A/G) have been used for suppressing primer-dimer structure formation during nucleic acid amplification. However, such constrained-randomized primers impart considerable bias in nucleic acid amplification reaction in terms of sequence coverage. Such primers are also of limited use for sequence-non-specific or sequence-non-biased nucleic acid amplification reactions (e.g., amplification of whole-genome, or amplification of a nucleic acid with unknown sequence). Thus, there exists a need for developing efficient nucleic acid amplification methods that have lower bias in terms of sequence coverage, and have lower levels of non-specific, background amplification. Development of primers that reduce primer-primer interaction, and can support nucleic acid amplification without sequence bias is also needed.